CN108685884A - A kind of hydroxytyrosol joint plumbagin is applied in immunological regulation and antitumor drug - Google Patents

Abstract

The invention discloses hydroxytyrosol joint plumbagins to be applied in immunological regulation and antitumor drug, including hydroxytyrosol and plumbagin, the hydroxytyrosol can improve effect of the plumbagin in terms of immunological regulation and antitumor drug in preparing immunological regulation and antitumor drug, the plumbagin can improve effect of the hydroxytyrosol in terms of immunological regulation and antitumor drug in preparing immunological regulation and antitumor drug, the plumbagin is a kind of anthraquinone analog compound extracted from Chinese medicine Plumbago zeylanica root, the hydroxytyrosol is the active ingredient of olive oil and plurality of Chinese.Hydroxytyrosol joint plumbagin is applied in immunological regulation and antitumor drug, hydroxytyrosol and plumbagin are used in combination, cooperative synergism effect may be implemented, significantly improve antitumous effect, hydroxytyrosol joint plumbagin can also significantly increase active o content in tumour cell, inducing apoptosis of tumour cell.

Description

A kind of hydroxytyrosol joint plumbagin is applied in immunological regulation and antitumor drug

Technical field

The present invention relates to field of medical technology, specially hydroxytyrosol combines plumbagin in immunological regulation and antineoplastic It is applied in object.

Background technology

Cancer is one of the main disease of current puzzlement human health, and the data announced according to the World Health Organization are shown, China increases cancer number of cases newly and death toll is in first, and wherein cancer of pancreas grade malignancy is high, American Cancer Society 2015 Year statistical result showed, cancer of pancreas five year survival rate find new safe and effective cancer treatment drugs less than 7% Hot and difficult issue through being studied as therapeutic field of tumor.Immuno Suppressive Therapy tumour is the hot spot studied now, due to tumour The inhibiting effect of microenvironment limits phagocytosis and killing of the immunocyte to tumour cell, has a large amount of marrow in tumor tissues The inhibitory cells and M2 type macrophages in source, inhibitory cells can be by promoting inflammation, promoting Tumor Angiongesis and obtaining Property immune response is obtained to promote tumour growth, inhibits activation and the proliferation of CD4+T cells and CD8+T cells, while tumour micro-loop Border inducing macrophage M2 types polarization reduces phagocytosis and killing to tumour cell, and the M1 types polarization of drug-induced macrophage can To promote phagocytosis and killing to tumour cell.Before immunomodulator and cell therapy use in conjunction show certain application Scape, research shows that inhibiting inhibitory cells cell proportion in tumor tissues to dramatically increase CAR-T to meat using all-trans retinoic acid The killing of oncocyte, in addition PD-L1 antibody can significantly increase CAR-T cell therapy curative effects, and CD47 antibody and Anthraquinones medicine Object can significantly increase phagocytosis of the macrophage to pancreatic cancer cell.

Plumbagin is a kind of anthraquinone analog compound extracted from Chinese medicine Plumbago zeylanica root, with clinical First-line chemotherapy medicine Ah mould Element has similar chemical structure, research shows that it is included in kinds of tumors model system in pancreatic cancer models, all shows wide The vitro cytotoxicity of spectrum and the effect of internal antitumor activity.Hydroxytyrosol is olive oil and plurality of Chinese (the wide windproof, fruit of glossy privet With plantain seed etc.) active ingredient, Mediterranean diet substantially reduces the incidence of local angiocardiopathy and cancer, wherein olive Fry dried food ingredients waves main function.

Hydroxytyrosol is a kind of compound containing catechol structure, has very strong antioxidant activity, while hydroxyl Tyrosol is that the main metabolites toxicity of dopamine in human body is very small, research shows that the hydroxytyrosol of large dosage can increase carefully Intracellular activity oxygen level kills colon cancer cell, and all there are one general character in most of anti-cancer therapies, are exactly reactive oxygen species Presence, radiotherapy also will produce reactive oxygen species in fact, and cell within a cell is increased clinically by the vitamin C of application large dosage Interior reactive oxygen species kill tumor stem cell.The natural materials of active oxygen are generated, it is at low cost and safe, it being greatly reduced The side effect brought is treated, there is wide potential applicability in clinical practice.But at present the antitumaous effect of hydroxytyrosol and plumbagin and Definite effect mechanism is still indefinite, limits its application in anti-tumor aspect, therefore we have proposed hydroxytyrosol joint is white Hua Dansu is applied in immunological regulation and antitumor drug.

Invention content

The purpose of the present invention is to provide hydroxytyrosol joint plumbagins to be applied in immunological regulation and antitumor drug, To solve the problems mentioned in the above background technology.

To achieve the above object, the present invention provides the following technical solutions:Hydroxytyrosol combines plumbagin in immunological regulation It is applied in antitumor drug, including hydroxytyrosol and plumbagin, the hydroxytyrosol is preparing immunological regulation and antitumor Effect of the plumbagin in terms of immunological regulation and antitumor drug can be improved in drug, the plumbagin is immune in preparation It adjusts and can improve effect of the hydroxytyrosol in terms of immunological regulation and antitumor drug, the Plumbago zeylanica in antitumor drug Element is a kind of anthraquinone analog compound extracted from Chinese medicine Plumbago zeylanica root, and the hydroxytyrosol is having for olive oil and plurality of Chinese Imitate ingredient.

Preferably, the hydroxytyrosol or plumbagin include its pharmaceutical salt with glucosides, selectively replace it is similar The derivative or the two and its derivative that the combination of object either one or more aforesaid compounds both further includes are pharmaceutically The solvate of acceptable salt or salt.

Preferably, the tumour is solid tumor.

Preferably, the ratio that the hydroxytyrosol and plumbagin add in drug is 100-300:1.

Preferably, the plurality of Chinese includes the wide windproof, fruit of glossy privet and plantain seed.

Compared with prior art, the beneficial effects of the invention are as follows:The hydroxytyrosol combine plumbagin in immunological regulation and It is applied in antitumor drug, hydroxytyrosol can improve cancer of pancreas cancer cell to plumbagin sensibility, and may be implemented to cooperate with The effect of synergy realizes that significantly improving for antitumous effect, hydroxytyrosol joint plumbagin can significantly increase tumour cell The content of middle active oxygen, inducing apoptosis of tumour cell, the application antitumor to hydroxytyrosol and plumbagin drug combination have Significant meaning, while the present invention specifies that in the pancreatic cancer models of Panc-02 mouse original position, hydroxytyrosol can significantly inhibit The ratio of the inhibitory cells of derived from bone marrow in tumor-bearing mice tumor tissues raises simultaneously M1 types macrophage in tumor tissues Ratio, it is also expressly that in the pancreatic cancer models of Panc-02 mouse original position, plumbagin can significantly inhibit tumor-bearing mice tumor group The ratio of the inhibitory cells of middle derived from bone marrow is knitted, while can significantly increase CD4+T cells and CD8+T cells in tumor tissues The ratio of subgroup.

Description of the drawings

Fig. 1 is the generation that hydroxytyrosol and plumbagin use in conjunction increase ROS in pancreatic cancer cell;

Fig. 2 is the apoptosis that hydroxytyrosol and plumbagin use in conjunction promote pancreatic cancer cell;

Fig. 3 is the inhibiting effect of hydroxytyrosol and plumbagin combination therapy to mouse original position pancreatic cancer growth;

Fig. 4 is influence of the hydroxytyrosol to inhibitory cells ratio in cancer of pancreas mouse tumor tissue;

Fig. 5 is influence of the hydroxytyrosol to M1 type macrophage ratios in cancer of pancreas mouse tumor tissue;

Fig. 6 is influence of the plumbagin to inhibitory cells ratio in cancer of pancreas mouse tumor tissue;

Fig. 7 is shadow of the plumbagin to CD4+T cells and CD8+T cell subsets ratios in cancer of pancreas mouse tumor tissue It rings;

Fig. 8 be hydroxytyrosol and plumbagin combination therapy to inhibitory cells in tumor tissues, M1 types macrophage and The influence of CD4+T cells and CD8+T cell subsets ratios;

Fig. 9 is plumbagin and hydroxytyrosol in vitro to the influence of macrophage phagocytic tumour cell.

Specific implementation mode

Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation describes, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other Embodiment shall fall within the protection scope of the present invention.

- 9 are please referred to Fig.1, the present invention provides a kind of technical solution:Hydroxytyrosol is combined plumbagin in immunological regulation and is resisted It is applied in tumour medicine, including hydroxytyrosol and plumbagin, the hydroxytyrosol or plumbagin include its pharmaceutical salt With the combination of glucosides, the analog selectively replaced or one or more aforesaid compounds, the derivative for both further including or Both persons and its derivative pharmaceutically solvate of acceptable salt or salt, the hydroxytyrosol and plumbagin are in drug The ratio of middle addition is 100-300:1, the hydroxytyrosol can improve white flower in preparing immunological regulation and antitumor drug Effect of the red element in terms of immunological regulation and antitumor drug, the plumbagin is in preparing immunological regulation and antitumor drug Effect of the hydroxytyrosol in terms of immunological regulation and antitumor drug can be improved, the plumbagin is from Chinese medicine Plumbago zeylanica root A kind of anthraquinone analog compound of middle extraction, the hydroxytyrosol are the active ingredient of olive oil and plurality of Chinese, it is described it is a variety of in Medicine includes the wide windproof, fruit of glossy privet and plantain seed, and the tumour is solid tumor, and experimental method of the invention is as follows:

Experiment one,

Experiment 1:Hydroxytyrosol and plumbagin use in conjunction increase the generation of ROS in pancreatic cancer cell

1, experiment material

(1) drug:Hydroxytyrosol, plumbagin, active oxygen probe DCFDA.

(2) cell:Mouse pancreas cancer Panc-02 cells.

2, experimental procedure

(1) experiment packet:Experiment is divided into four groups, control group (Cont), hydroxytyrosol group (HT), plumbagin group (PLB) With hydroxytyrosol+plumbagin group (HT+PLB).

(2) specific experimental method is as follows:After the Panc-02 cells of exponential phase are digested with pancreatin, 4 × 105 cells are taken Uniformly be taped against in six orifice plates, according to experiment packet agent-feeding treatment for 24 hours after, be added DCFDA probes, 37 DEG C be incubated 30min after, digestion Cell passes through flow cytomery intracellular activity oxygen content.

3, experimental result

After experimental result using hydroxytyrosol (150 μM) or the single medicine of (5 μM) of plumbagin as shown in Figure 1, handled, pancreas The generation of ROS obviously increases after the generation of ROS increased in cancer cell, but the two is used in combination.

Experiment two,

Experiment 2:Hydroxytyrosol and plumbagin use in conjunction increase the apoptosis of pancreatic cancer cell

1, experiment material

(1) drug:Hydroxytyrosol and plumbagin.

(2) cell:Mouse pancreas cancer Panc-02 cells.

(3) apoptosis kit.

2, experimental procedure

(1) experiment packet:Experiment is divided into four groups, control group (Cont), hydroxytyrosol group (HT), plumbagin group (PLB) With hydroxytyrosol+plumbagin group (HT+PLB).

(2) the specific method is as follows:After the Panc-02 cells of exponential phase are digested with pancreatin, take 4 × 105 cells uniform Be taped against in six orifice plates, according to experiment packet agent-feeding treatment for 24 hours after, vitellophag, according to the method for apoptosis detection kit handle Cell, Annexin V and PI dyeing pass through flow cytomery Apoptosis situation after completing.

3, experimental result

Experimental result is as shown in Figure 2 A, and the double positives of Annexin V and PI dyeing are non-viable apoptotic cell ratio, use hydroxyl After tyrosol (150 μM) or the single medicine processing of (5 μM) of plumbagin, apoptosis of pancreatic cancer cell ratio increased, but the two joins Apoptosis ratio obviously increases after closing use, such as Fig. 2 B.

Experiment three,

Experiment 3:Hydroxytyrosol and the growth of plumbagin mice pancreatic carcinoma in situ and the influence of tumor microenvironment

1, experiment material

(1) drug:Hydroxytyrosol, plumbagin.

(2) cell:Mouse pancreas cancer Panc-02 cells.

(3) streaming antibody:Anti-mouse CD4 PE,Anti-mouse CD8 APC,PeCy7 Anti-mouse Ly6G PE,Anti-mouse MHC classⅡ PE,Anti-mouse CD11b FITC,Anti-mouse F4/80 APC With Anti-mouse CD3 FITC.

(4) experimental animal:C57BL/6 mouse, it is female, 6-8 weeks, SPF grades.

2, experimental procedure

(1) experiment packet:Experiment is divided into four groups, control group (Cont), hydroxytyrosol group (HT), plumbagin group (PLB) With hydroxytyrosol+plumbagin group (HT+PLB)

(2) structure of orthotopic mouse pancreatic cancer models:

1) inoculation Panc-02 cells are in culture dish, and with the DMEM culture solutions containing 10% fetal calf serum, culture to logarithm is given birth to For a long time;After pancreas digestion, PBS is washed 2 times, and it is 2 × 107/ml that cell concentration is adjusted after being resuspended with PBS;

2) ketalar (150mg/kg) and xylazine hydrochloride (65ug/kg) anesthetized mice is injected in mouse peritoneal, 75% chronic ethanol treated mice abdomen carries out disinfection, and mouse left abdomen opens the osculum of about 1cm, the visible pancreas around spleen, uses The cell that the insulin syringe of 1cm injects 50 μ L in tail of pancreas portion, sutures after gently putting back to abdominal cavity, revives naturally;

3) third day starts to be administered after being inoculated with, and is injected intraperitoneally, dosage is hydroxytyrosol 200mg/kg, plumbagin 1mg/kg draws materials after treating 10 days, 14 days altogether, takes tumour, and weigh tumor weight.

4) tumor tissues immunocyte is detached:

1. tumor tissues are placed in 50mL centrifuge tubes, the digestive juice (clostridiopetidase A+hyaluronic acid that 5mL has been configured is added Enzyme), and surgically cut and tumor tissues are cut into the fragment for being about 1mm to diameter, then centrifuge tube is placed in 37 DEG C of water-baths Vibrate 60min;

2. postdigestive tissue is placed on the stainless steel mesh of 200 mesh, appropriate precooling PBS is added, uses syringe piston Backside is lightly ground, it is to be ground completely after, collect filtrate;Filtrate 4 DEG C, after centrifuging 5min, abandons supernatant, uses 5mL through 2000rpm The 40%percoll liquid configured is resuspended;

3. taking 15mL centrifuge tubes, the 80%percoll liquid that 3mL has been configured is added, then cell suspension is slowly added to On 80%percoll liquid levels;600g, room temperature accelerate 5, and deceleration 0 centrifuges 30min;

4. steadily taking out centrifuge tube, gos deep into liquid level gentle aspiration tunica albuginea layer with Dispette, be transferred to the centrifugation of 15mL The PBS of 3 times of volumes or more is added in Guan Zhong;2000rpm, centrifuges 5min by 4 DEG C, and precipitation is tumor tissues mononuclearcell.

5) flow cytometer detection immunocyte subgroup:

1. tumor tissue cell 100ul obtained as above is added in each detection pipe;

2. carrying out fluorescent marker dyeing according to following scheme.

A) CD3 FITC, CD8 APC, CD4 PE (label CD4 and cd8 t cell);

B) CD11b FITC, Ly6G PE (label MDSCs cells);

C) CD11b FITC, F4/80 APC, II PE of MHC class (label M1 types macrophage).

After 3. 4 DEG C are protected from light dyeing 40min, PBS washes 2 times, flow cytomery.

3, experimental result

As shown in figure 3, compared with model group, the weight of hydroxytyrosol group and plumbagin group tumour all decreases, and The weight of drug combination group tumour further decreases (* P&lt compared with independent medication group;0.05,#P<0.01), illustrate drug combination Have the function of synergy in terms of anti-pancreatic cancer, ROS increases and apoptosis ratio in tumour cell are induced with external drug combination The increase result of example is consistent.In order to further probe into its mechanism, we detect each group to immunocyte ratio in tumor tissues respectively The influence of example, the experimental results showed that, hydroxytyrosol is not notable to CD4+T cells and CD8+T cell subsets scale effects, but its The ratio (Fig. 4) of MDSCs cells in tumor tissues can be significantly reduced and increase the ratio (Fig. 5) of M1 type macrophages;White flower Influence of the red element to M1 types macrophage ratio in tumor tissues be not notable, but it can equally reduce the ratio of inhibitory cells Example (Fig. 6);In addition, plumbagin can significantly increase the ratio (figure of CD4+T cells and CD8+T cell subsets in tumor tissues 7);The ratio of inhibitory cells is remarkably decreased in drug combination group tumor tissues, and M1 types macrophage, CD4+T cells and CD8 The ratio of+T cell subgroup significantly rises (Fig. 8).Illustrate that drug combination can significantly reduce immunosuppressant cell ratio and raising Immunologic cytotoxicity cell proportion (M1, CD4+T and CD8+T), drug combination also have synergistic in terms of improving tumor microenvironment Effect.

Experiment four,

Experiment 4:Plumbagin and hydroxytyrosol are in vitro to the influence of macrophage phagocytic tumour cell

Hydroxytyrosol can significantly increase the ratio of M1 types macrophage in tumor tissues, and research report M1 types macrophage is to swollen The phagocytosis and killing ability enhancing of oncocyte, in addition research shows that Anthraquinone can promote calprotectin to cell surface Transport promotes phagocytosis of the macrophage to target cell, therefore we check whether hydroxytyrosol and Plumbago zeylanica drug combination promote The phagocytosis of Macrophages For Tumor kills.

1, experiment material

(1) drug:Hydroxytyrosol, plumbagin, sodium thioglycollate and CFSE.

(2) cell:Mouse pancreas cancer Panc-02 cells, mouse RAW264.7 macrophages and the primary macrophage of mouse peritoneal Cell.

(3) streaming antibody:F4/80 APC.

(4) experimental animal:C57BL/6 mouse, it is female, 6-8 weeks, SPF grades.

2, experimental procedure

(1) experiment packet:Experiment is divided into four groups, control group (Cont), hydroxytyrosol group (HT), plumbagin group (PLB) With hydroxytyrosol+plumbagin group (HT+PLB).

(2) the specific method is as follows:

1) separation of mouse peritoneal primary macrophage

C57BL/6 mouse are randomly selected, 2ml/ mouse of 3% sodium thioglycollate is injected intraperitoneally testing first three day.It will Mouse cervical dislocation is put to death, and as on dissection plate, is extracted 5mL with syringe and is injected abdominal cavity containing dual anti-RPMI1640 culture mediums, Abdomen 1-2min is gently rubbed with silk tops, extracts peritoneal fluid with syringe, centrifugation 5min (1000r/min) abandons supernatant, 2 are washed with PBS Time.It is resuspended again with the RPMI-1640 culture solutions containing 10% bovine serum, sets 37 DEG C, 5%CO2 incubator culture 2h, attached cell As peritoneal macrophage, the detection for tumour cell phagocytosis experiment.

2) pancreatic cancer marker and external phagocytosis experiment

After the Panc-02 cells of exponential phase are digested with pancreatin, 4 × 105 cells is taken uniformly to be taped against in six orifice plates, according to Experiment packet agent-feeding treatment for 24 hours after, vitellophag is added final concentration of 5 μM of CFSE and tumour cell is marked, and 37 DEG C incubate After educating 10min, fetal calf serum is added and terminates reaction, 2 times, 37 DEG C and primary abdominal cavity macrophage are washed with the culture medium of the DMEM of serum-free Cell or RAW264.7 macrophages are incubated 2h altogether, and the F4/80 antibody of fluorescent marker marks macrophage, flow cytomery The ratio of phagocyte.

3, experimental result

Experimental result is as shown in Figure 9 A, and hydroxytyrosol does not influence phagocytosis of the RAW264.7 macrophages to tumour;In vain It spends red element that can remarkably promote the phagocytosis of RAW264.7 Macrophages For Tumors, can also be remarkably promoted after drug combination The phagocytosis of RAW264.7 Macrophages For Tumors;As shown in Figure 9 B, primary after plumbagin and hydroxytyrosol drug combination The ratio of macrophage phagocytic tumour cell equally obviously increases, and illustrates that drug combination is promoting Macrophages For Tumor Also there is synergy in terms of phagocytosis.

In conclusion hydroxytyrosol and plumbagin are used in combination, synergistic effect may be implemented, significantly improve Antitumor effect, hydroxytyrosol joint plumbagin can significantly increase the content of active oxygen in tumour cell, induce tumour Apoptosis, the application antitumor to hydroxytyrosol and plumbagin drug combination have significant meaning.

It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of variations, modification, replace And modification, the scope of the present invention is defined by the appended.

Claims (5)

1. hydroxytyrosol joint plumbagin is applied in immunological regulation and antitumor drug, including hydroxytyrosol and Plumbago zeylanica Element, it is characterised in that:The hydroxytyrosol can improve plumbagin immune in preparing immunological regulation and antitumor drug Effect in terms of adjusting with antitumor drug, the plumbagin can improve hydroxyl in preparing immunological regulation and antitumor drug Effect of the base tyrosol in terms of immunological regulation and antitumor drug, the plumbagin are one extracted from Chinese medicine Plumbago zeylanica root Class anthraquinone analog compound, the hydroxytyrosol are the active ingredient of olive oil and plurality of Chinese.

2. hydroxytyrosol joint plumbagin according to claim 1 is applied in immunological regulation and antitumor drug, It is characterized in that:The hydroxytyrosol or plumbagin include its pharmaceutical salt and glucosides, the analog selectively replaced or The combination of one or more aforesaid compounds, the derivative for both further including or the two and its derivative are pharmaceutically subjected to Salt or salt solvate.

3. hydroxytyrosol joint plumbagin according to claim 1 is applied in immunological regulation and antitumor drug, It is characterized in that:The tumour is solid tumor.

4. hydroxytyrosol joint plumbagin according to claim 1 is applied in immunological regulation and antitumor drug, It is characterized in that:The ratio that the hydroxytyrosol and plumbagin add in drug is 100-300:1.

5. hydroxytyrosol joint plumbagin according to claim 1 is applied in immunological regulation and antitumor drug, It is characterized in that:The plurality of Chinese includes the wide windproof, fruit of glossy privet and plantain seed.